doi:10.1006/jmbi.2000.3844availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (2000) 300, 353±362 A TOPRIM Domain in the Crystal Structure of the Catalytic Core of Escherichia coli Primase Confirms a Structural Link to DNA Topoisomerases MarjetkaPodobnik1,3,PeterMcInerney2,MikeO'Donnell2 andJohnKuriyan1* 1Laboratories of Molecular Primases synthesize short RNA strands on single-stranded DNA tem- Biophysics and plates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases. We present the crystal structure of the 2Laboratory of DNA catalytic unit of a primase enzyme, that of a 320 residue fragment of Replication, Howard Hughes Escherichia coli primase, determined at 2.9 AÊ resolution. Central to the cat- Medical Institute, The alytic unit is a TOPRIM domain that is strikingly similar in its structure Rockefeller University, New to that of corresponding domains in DNA topoisomerases, but is unre- York, NY 10021, USA lated to the catalytic centers of other DNA or RNA polymerases. The cat- 3Department of Biochemistry alytic domain of primase is crescent-shaped, and the concave face of the and Molecular Biology, Jozef crescent is predicted to accommodate about 10 base-pairs of RNA-DNA Stefan Institute, Slovenia duplex in a loose interaction, thereby limiting processivity. # 2000 Academic Press Keywords: DNA replication; RNA-polymerase; primase; TOPRIM; *Corresponding author topoisomerase Introduction 65.5 kDa protein (581 residues) that contains three functionally distinct regions that are separ- able by proteolysis. The N-terminal region None of the known DNA polymerases is able to initiate DNA synthesis by using single-stranded (12 kDa, residues 1-110) contains a zinc-binding DNA as templates. Instead, DNA replication relies domain that is required for primase function, on a variety of mechanisms to generate short pri- potentially because of a role in recognizing single- mer regions, typically RNA-DNA hybrid duplexes, stranded DNA. The crystal structure of this that then serve as initiation sites for DNA synthesis domainhasbeendetermined(Pan&Wigley, by DNA polymerases. During chromosomal repli- 2000).Acentralcatalyticdomain(36kDa,resi- cation, in particular, the repeated generation of dues 111-433) is responsible for the catalysis of RNA primers required for the replication of lag- RNA synthesis, and a C-terminal fragment (resi- ging strands, is brought about by DNA-dependent dues 434-581) is involved in interactions between RNA polymerases, known as primases. theprimaseandthehelicase(Tougu&Marians, The prototypical bacterial primase is the enzyme 1996;Touguetal.,1994).Primaseactivityrequires encoded by the dnaG gene of Escherichia coli both the N-terminal zinc-binding domain and the (Kornberg&Baker,1991).Allknownbacterialpri- central catalytic domain. mases, as well as primases from several bacterio- Bacterial and bacteriophage primases of the phages, are homologous to E. coli primase. DnaG family have no apparent relationship to Referred to collectively as DnaG-type primases, eukaryotic and archaebacterial primases, or to any these are often closely associated with DNA heli- otherRNAorDNApolymerases(Aravindetal., cases in mobile assemblies known as primosomes, 1998;Leipeetal.,1999).Unexpectedly,iterative which track the moving replication fork sequence pro®le searches using the program PSI- (McMacken&Kornberg,1978).E.coliprimaseisa BLAST and the E. coli primase sequence as a start- ing input revealed a potential structural relation- ship between a central region (100 residues) of Abbreviations used: MAD, multiwavelength anomalous dispersion; TEV, tobacco etch virus; NCS, the catalytic domain of DnaG-type primases and non-crystallographic symmetry. otherwise unrelated proteins such as certain DNA E-mail address of the corresponding author: topoisomerases, several nucleases and proteins [email protected] involvedinrecombinationalrepair(Aravindetal., 0022-2836/00/020353±10 $35.00/0 # 2000 Academic Press 354 Structure of E. coli Primase Catalytic Core 1998).ThissharedregionwasnamedtheTOPRIM stranded DNA template and the nascent RNA- domain (for topoisomerase/primase). The level of DNA duplex with the primase. Our conclusions sequence similarity upon which the presence of are in general agreement with an independent TOPRIM domains in bacterial primases was postu- report of the structure of the catalytic core of E. coli lated is relatively low. The TOPRIM domains in primase that was published at the same time as the primases are smaller than those in topoisomerases submissionofthispaper(Kecketal.,2000). (80 residues instead of 120 residues), and only 10 % of the residues in the TOPRIM domains of Results and Discussion known structure (all of which are in topoisome- rases) are preserved identically in any one of the General features of the structure primase sequences. The boundaries of the catalytic core of E. coli pri- We have used X-ray crystallography to deter- mase were de®ned by subjecting the full-length mine the crystal structure of the catalytic core of protein to limited proteolytic digestion with tryp- E. coli primase. Embedded within the catalytic sin,asdescribed(Touguetal.,1994),andcharacter- domain is a TOPRIM fold that is similar to that izing the resulting fragments by N-terminal seen in the topoisomerases, con®rming the striking sequencingandmassspectrometry(Figure1(a); conservation of this domain among functionally data not shown). A stable fragment comprising different replication proteins. The structural simi- residues 111-429 was identi®ed, corresponding to larity between the TOPRIM domains of primase thecentralcatalyticregionofprimase(Mustaev& andtopoisomeraseVI(Nicholsetal.,1999)allows Godson,1995).ThestructureofE.coliprimasecat- us to identify a metal-binding site in the primase alytic core was determined at 2.9 AÊ resolution catalytic domain, and thereby to locate the likely using multiwavelength anomalous dispersion site for nucleotide addition to a growing primer (MAD) and a single crystal of selenomethionyl chain. Combined with analysis of surface features substitutedprotein(Table1). of the primase this has enabled a hypothesis to be The crystallographic asymmetric unit contains generated regarding the interaction of the single- ®ve molecules of primase, which are arranged in a Figure 1. The structure of the E. coli primase catalytic domain. (a) A diagram of the domain organization of full- length primase. The residue numbers corresponding to the domain boundaries in the E. coli protein are indicated. (b) Ribbon representation of the structure of the primase catalytic domain. The orientation shown here is similar to that used in most of the Figures in the paper. The b strands are marked with the red numbers (1-12) and a-helices with black letters (A-N). The circles indicate the boundaries of the TOPRIM sub-domain. Structure of E. coli Primase Catalytic Core 355 Table 1. Data collection and re®nement statistics A. MAD phasing Energies (eV) No. of reflections (total/unique) Completeness (%) Rsym l1 12,570 273,234/40,874 97.6 (90.8) 5.8 (20.7) l2 12,660 276,321/40,801 97.9 (93.2) 5.8 (21.2) l3 12,664 279,034/40,836 98.0 (93.7) 7.5 (23.4) l4 12,845 279,399/40,704 98.5 (97.4) 6.2 (22.2) Mean overall figure of merit (15.0 AÊ -2.9 AÊ ) (centric/acentric)0.51/0.58 B. Refinement and stereochemical statistics R-value (%) 21.9 Free R-value (%) 27.6 AverageB-factorsa(AÊ2) Main-chain 46.81 Side-chains 50.95 Whole 48.82 RMS deviations Bonds (AÊ ) 0.0114 Angles (deg.) 1.414 Rsym 100 Â ÆjI hIij/ÆI, where I is the integrated intensity of a given re¯ection. For Rsym and completeness, numbers in parentheses refer to data in the highest resolution shell. Figure of merit hjÆP(a)eia/ÆP(a)ji, where a is the phase and P(a) is the phase probability distribution. R-value ÆjFp Fp(calc)j/ÆFp, where Fp is the structure factor amplitude. The free R-value is the R-value for a 10 % subset of the data that was not included in the crystallographic re®nement. a Average B-factors for the ®ve molecules in the asymmetric unit. helical spiral. The ®ve molecules differ slightly in their molecular architecture and their enzymatic their inter-domain orientation, but the structures of mechanisms(Limaetal.,1994;Bergeretal.,1996; the individual domains are virtually unchanged. Nicholsetal.,1999).Nevertheless, the three-dimen- This assembly is unlikely to be of physiological sig- sional structures of both type IA and type II topoi- ni®cance, but it is of interest, since it originates somerases have a small core region in common, from the interaction of an arginine residue side- which corresponds to theTOPRIMdomain chain (residue 299) in one protein with the putative (Aravindetal., 1998;Bergeretal.,1998).The fold metal-binding site of another. This arrangement of the TOPRIM domains in these proteins positions the guanidium group of the arginine resi- resembles a Rossmann-like nucleotide-binding due such that it mimics the metal observed in the fold, with a central b-sheet formed by four parallel TOPRIM domain of topoisomerase VI (not shown). b-strands, ¯anked by three a-helices. Both type IA Our attempts to soak nucleotides into this crystal and type II topoisomerases contain a tyrosine resi- form have been unsuccessful, almost certainly due that forms a covalent linkage with DNA because the addition of divalent metal ions will during one stage of the topoisomerase reaction disrupt this interaction between molecules in the cycle. This tyrosine residue is not part of the crystal.